BACKGROUND: MS-associated autonomic dysfunction (AD) in multiple sclerosis (MS) is poorly understood and the best method for its detection unestablished. We compared classical Ewing battery and newer methods as heart rate variability (HRV) and spontaneous baroreflex sensibility (BRS) to detect AD in MS and related them to central autonomic network (CAN) lesions. METHODS: We enrolled 20 relapsing-remitting MS patients, median age of 36 (interquartile range 32-46) years, disease duration of 5.5 (2.2-6.8) years, Expanded Disability Status Scale (EDSS) score of 1.0 (1.0-1.5) and 20 age- and gender-matched healthy controls. We assessed Ewing battery and spontaneous HRV and BRS. CAN involvement was evaluated by magnetic resonance imaging. RESULTS: HRV showed both parasympathetic and sympathetic significant impairment in MS (p<0.05). From Ewing battery only isometric test was significantly decreased in MS (p=0.006). Disease duration and severity, lesion burden and CAN involvement were not correlated with laboratorial parameters. CONCLUSIONS: Our MS cohort had both sympathetic and parasympathetic dysfunction independently from disease duration, neurological deficits and lesion burden or CAN involvement. HRV analysis maybe more useful than classical Ewing battery to screen AD.